In spite of massive studies on protein biosynthesis, a full understanding of this process still awaits a reliable model. The long term goal of this project is to illuminate the molecular mechanism of protein biosynthesis. The immediate objective is to elucidate the three-dimensional structure of ribosomes, the site of this process. Diffraction methods provide the only techniques for direct determination of structures. Usage of these methods is dependent on the availability of crystalline material. Experimental procedures for production of crystals and sheets of ribosomal particles have been developed, and unique systems which can be studied at relatively high resolution are available. Crystallographic studies, using an intense synchrotron X-ray beam at cryotemperature, will be carried out on crystals of large and small ribosomal subunits from eu- and halobacteria. Complexes of ribosomal particles with tRNA and nascent protein chains, mutated particles, missing a ribosomal protein, and modified particles, whose free -SH groups have been covalently bound to heavy-atom clusters will be studied. The crystallographic work will be supported by biochemistry, electron microscopy and neutron diffraction. Ribosomal particles to which functional probes (e.g. cDNA or tRNA) are attracted will also be studied. Comparisons of the structures of probed and native particles should indicate the locations of selected steps in protein biosynthesis. In addition, 705 particles, "frozen" in distinct functional states, will be obtained by fine tuning of homogeneous populations of these particles. Messenger RNA, coding for the synthetic sequences as well as for naturally occurring proteins, will be used for in vitro biosynthesis. The length of the newly synthesized oligopeptide will be controlled by omitting a selected amino acid from the reaction mixture. The extent of binding of the various nascent chains to the ribosomes will be measured. The firmly bound complexes will be crystallized. The significance of our research plan stems from the fundamental value of increased understanding of a basic life process, as well as from its potentials applicative aspect. Thus, elucidation of the effect of many antibiotics at the molecular level will become possible once the structure of ribosomes is known since these act on ribosomes and block various steps of protein biosynthesis. Furthermore, our studies may provide the basic principles for design of powerful and efficient therapeutic agents. Also, it is conceivable that the detailed knowledge of the pathway of protein biosynthesis may shed a light on abnormal and pathological deviations. Last, but not least, our studies are bound to contribute to the development of sophisticated crystallographic experimental methods.